EP2984072A1 - Synthetische analoga von xanthohumol - Google Patents

Synthetische analoga von xanthohumol

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EP2984072A1
EP2984072A1 EP14727911.1A EP14727911A EP2984072A1 EP 2984072 A1 EP2984072 A1 EP 2984072A1 EP 14727911 A EP14727911 A EP 14727911A EP 2984072 A1 EP2984072 A1 EP 2984072A1
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Prior art keywords
phenyl
enyl
methoxy
methyl
alkyl
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French (fr)
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EP2984072B1 (de
Inventor
Armando Rossello
Elisa Nuti
Elisabetta Orlandini
Susanna Nencetti
Adriana Albini
Anna Rita CANTELMO
Desiree BARTOLINI
Douglas M. Noonan
Cristina GALLO
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Universita di Pisa
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Azienda Ospedaliera Arcispedale Santa Maria Nuova/ Irccs Istituto In Tecnologie Avanzate E Modelli Assistenziali In Oncologia
Universita di Pisa
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    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/44—Radicals substituted by doubly-bound oxygen, sulfur, or nitrogen atoms, or by two such atoms singly-bound to the same carbon atom
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    • C07C205/57—Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by carboxyl groups having nitro groups and carboxyl groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton
    • C07C205/61—Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by carboxyl groups having nitro groups and carboxyl groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton the carbon skeleton being further substituted by doubly-bound oxygen atoms
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
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    • C07C233/30—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by doubly-bound oxygen atoms
    • C07C233/33—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by doubly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/45—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups
    • C07C233/53—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring
    • C07C233/55—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring having the carbon atom of the carboxamide group bound to a carbon atom of an unsaturated carbon skeleton
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/01—Sulfonamides having sulfur atoms of sulfonamide groups bound to acyclic carbon atoms
    • C07C311/02—Sulfonamides having sulfur atoms of sulfonamide groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton
    • C07C311/08—Sulfonamides having sulfur atoms of sulfonamide groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton having the nitrogen atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/22—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound oxygen atoms
    • C07C311/29—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound oxygen atoms having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring
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    • C07C311/50—Compounds containing any of the groups, X being a hetero atom, Y being any atom
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    • C07C49/76—Ketones containing a keto group bound to a six-membered aromatic ring
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    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/61—Halogen atoms or nitro radicals
    • C—CHEMISTRY; METALLURGY
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4

Definitions

  • the present invention relates to novel synthetic analogues of xanthohumol (XN), a prenylated chalcone of natural origin present in hops ⁇ Humulus lupulus).
  • Natural compounds extracted from plants such as flavonoids, exhibit chemopreventive or therapeutic properties in various disorders.
  • the flavonoids contained in many foods and drinks commonly used for human consumption include compounds belonging to various structural classes, such as flavones, flavonols (quercetin), catechins, flavonones (naringenin), isoflavones and chalcones (xanthohumol).
  • Xanthohumol XN is the main chalcone contained in the female inflorescence of the hop plant ⁇ Humulus lupulus L. - Cannabaceae), and is extensively used in beer manufacturing processes for adding flavour and aroma and stabilising the froth. Beer, wherein XN is present in concentrations of about 0.96 mg/1 (1.95 ⁇ ), can represent one of the major sources of flavones in the diet.
  • XN possesses anti-invasive, anti-proliferative, anti-angiogenic, pro-apoptotic, anti-infective and inhibiting activities on the enzymes of cytochrome P450 involved in the metabolic activation of carcinogenesis.
  • the properties of XN also include antiinflammatory activity, exercised by reducing the production of nitric oxide (NO), a free radical involved in carcinogenesis and angiogenic processes [Zhao F, Nozawa H, Daikonnya A, Kondo K, Kitanaka S. Inhibitors of nitric oxide production from hops (Humulus lupulus L.). Biol Pharm Bull. 2003 Jan; 26(l):61-5].
  • NO nitric oxide
  • XN has an inductive effect on quinone reductase, which modulates the hepatic expression of the genes involved in the distribution, in the metabolism of thyroid hormones, and the metabolism of glucose and lipids, and an immunomodulating affect against skin aging [Dietz BM, Kang YH, Liu G, Eggler AL, Yao P, Chadwick LR, Pauli GF, Farnsworth NR, Mesecar AD, van Breemen RB, Bolton JL. Xanthohumol isolated from Humulus lupulus Inhibits menadione-induced DNA damage through induction of quinine reductase. Chem Res Toxicol. 2005 Aug; 18(8): 1296-305].
  • XN induces apoptosis in breast cancer cells by inducing cleavage of the PARP
  • MMP-2 gelatinase A
  • MMP-9 gelatinase A
  • TIMP-1 an MMP tissue inhibitor which forms a complex with pro- MMP9
  • the activity of MMP-2 is also strongly inhibited by XN (10 ⁇ ) [Philips N, Samuel M, Arena R, Chen YJ, Conte J, Natarajan P, Haas G, Gonzalez S.
  • XNC also known as dehydroxanthohumol, is a pyranochalcone whose principal activities are antifungal, anti-proliferative, anti- mutagenic and antioxidant. This compound, unlike XN, has a pyranose ring instead of the prenyl group on C-3', which involves loss of cytotoxic activity. Moreover, although XN- SEM presents the prenyl group as XN, it has no cytotoxic effect on fibrosarcoma cells.
  • XN also exhibits anti-angiogenic activity. Vascularisation of the tumour is necessary for its growth and metastatic spread. Inhibiting and delaying angiogenesis are therefore possible strategies for the treatment and prevention of cancer. Studies have been conducted on the changes induced by XN in the various cell populations most involved in tumour angiogenesis, namely endothelial cells. XN, at ⁇ concentrations, reduces in vitro the proliferation, invasive activity and ability of the endothelial cells to form a tubular network in extracellular matrix.
  • XN inhibits activation of NF- kB and phosphorylation of IkBa, preventing the translocation of NF-kB to the nucleus and the expression of pro-inflammatory genes [Monteiro R, Calhau C, Silva AO, Pinheiro-Silva S, Guerreiro S, Gartner F, Azevedo I, Soares R. Xanthohumol inhibits inflammatory factor production and angiogenesis in breast cancer xenografts. J Cell Biochem. 2008 Aug 1; 104(5): 1699-707].
  • XN also prevents angiogenesis and reduces tumour growth in assays conducted in vivo [Adriana Albini, Raffaella Dell' Eva, Nicoletta Ferrari; "Mechanisms of the antiangiogenic activity by the hop flavonoid xanthohumol: NF-kB and Akt as targets”; The FASEB Journal: 10.1096/fj.05-5128fje, 30 December 2005].
  • XN inhibits the phosphorylation of AKT, a serine/threonine kinase which is important in regulating cell migration and survival signals [Adriana Albini, Raffaella Dell'Eva, Nicoletta Ferrari; "Mechanisms of the antiangiogenic activity by the hop flavonoid xanthohumol: NF-kB and Akt as targets"; The FASEB Journal: 10.1096/fj.05-5128fje, 30 December 2005].
  • Endothelial cells activated by an angiogenic stimulus enter a proliferative state.
  • XN performs a potent cytotoxic activity against these cells at the dose of 10 ⁇ , whereas at lower concentrations it does not exhibit any significant effect, even after long exposure times. Cell death is only observed at concentrations exceeding 25 ⁇ . XN inhibits chemotaxis of the endothelial cells and invasion in vitro at low concentrations (5 ⁇ ), with complete inhibition at 10 ⁇ [Adriana Albini, Raffaella Dell'Eva, Nicoletta Ferrari; "Mechanisms of the antiangiogenic activity by the hop flavonoid xanthohumol: NF-kB and Akt as targets"; The FASEB Journal: 10.1096/fj.05-5128fje, 30 December 2005].
  • XN also acts directly on the activation of the NF-kB dependent kinase IkBa (IKK), interacting with the sulphydryl group of cysteine 179 of IKK. Through interaction with these cysteine groups, XN boosts its apoptotic action and suppresses the production of anti- apoptotic genes [Kuzlil B. Harikumar, Ajaikumar B. Kunnumakkara, Kwang S.
  • IKK NF-kB dependent kinase IkBa
  • Prostate cancer is the most common form of cancer diagnosed in men.
  • Deeb et al. have studied the effects of XN on hormone-sensitive and hormone-refractory prostate cancer.
  • the results demonstrate the inhibitory effect of XN on the growth of prostate cancer cell lines (40 ⁇ ) and the induction of apoptosis via the intrinsic pathway, which involves activation of pro-caspases -3, -8 and -9, mitochondrial depolarisation and the release of cytochrome C from the mitochondrion [Deeb D, Gao X, Jiang H, Arbab AS, Dulchavsky SA, Gautam SC.
  • XN also inhibits antiapoptotic proteins Akt, mTOR and NF-kB, suggesting that their inactivation is necessary for the induction of apoptosis [D. Deeb, X.
  • XN possesses other biopharmacological activities, such as antibacterial activity [Clarissa Gerhauser; "Broad spectrum antiinfective potential of xanthohumol from hop (Humulus lupulus L.) in comparison with activities of other hop constituents and xanthohumol metabolites”; Mol. Nutr. Food Res: 49, 827-831 (2005); Teuber M., Schmalreck A.F., "Membrane leakage in Bacillus subtilis 168 induced by the hop constituents lupulone, humulone, isohumulone and humulinic acid”; Arch.
  • XN inhibits the cytopathic effects induced by the HIV-1 virus, the production of viral antigen P24, and reverse transcriptase activity [Buckwold V.E., Wilson R.J., Nalca A., Beer B.B. et AL; "Antiviral activity of hop constituents against a series of DNA and RNA viruses”; Antiviral Research: 61, 57-62 (2004)].
  • XN and other constituents of hops exhibited inhibitory activity in vitro against a series of RNA and DNA viruses.
  • RNA virus BVDV bovine diarrhoea virus, an analogue of the hepatitis C virus, HCV
  • CMV cytomegalovirus
  • HSV-1 Herpes simplex type 1
  • HSV-2 Herpes simplex type 2
  • IXN is active at higher concentrations on all the species cited [Buckwold V. E., Wilson R.J., Nalca A., Beer B.B. et Al.; "Antiviral activity of hop constituents against a series of DNA and RNA viruses”; Antiviral Research: 61, 57-62 (2004)].
  • the BVDV and HCV viruses belong to the Flaviviridae family, and share homologous protein regions, such as virion structure, genome organisation and replication strategy [Buckwold V.E., Wilson R.J., Nalca A., Beer B.B. et AL; "Antiviral activity of hop constituents against a series of DNA and RNA viruses”; Antiviral Research: 61, 57-62 (2004)].
  • BVDV is often used as a surrogate model to evaluate potential compounds with antiviral activity on HCV.
  • XN can be used as a base for the development of novel anti-HCV compounds.
  • the combination of the two may therefore offer substantial benefits to individuals who are particularly sensitive to high doses of interferon [Ni Zhang, Zhengwen Liu, Qunying Han; "Xanthohumol enhances antiviral effect of interferon a-2b against bovine viral diarrhea virus, a surrogate of hepatitis C virus”; Phytomedicine: 17, 310-316 (2010)].
  • XN and 6- prenylnaringenin proved to be the most potent antifungal agents, inhibiting the growth of the dermatophytes T. mentagrophytes, T. rubrum and M. rouxianus more efficiently than griseofulvin, used as reference.
  • IXN is inactive.
  • C. albicans does not respond to XN or naringenin [Frolich S, Schubert C, Bienzle U, Jenett-Siems K. In vitro antiplasmodial activity of prenylated chalcone derivatives of hops (Humulus lupulus) and their interaction with haemin. J Antimicrob Chemother. 2005 Jun;55(6):883-7].
  • XN also exhibits antiprotozoal activities by acting on cell strains of chloroquine- sensitive and chloroquine-resistant plasmodia, by means of an mechanism action which is not yet clear [Frolich S., Schubert C, Bienzle U.; "In vitro antiplasmodial activity of prenylated chalcone derivates of hops (Humulus lupulus) and their interaction with haemin”; Journal of Antimicrobial Chemotherapy: 55, 883-887 (2005)].
  • XN Another pharmacological activity of XN is its anti-inflammatory activity.
  • Chronic neuroinflammation is a characteristic of neurodegenerative disorders, such as Parkinson's disease (PD), involving activation of the microglial cells in the substantia nigra, increased levels of pro-inflammatory cytokines in the striata and the substantia nigra, and activation of the pro-inflammatory NF-kB signalling pathway, [Lee IS, Lim J, Gal J, Kang JC, Kim HJ, Kang BY, Choi HJ.
  • Anti-inflammatory activity of xanthohumol involves heme oxygenase- 1 induction via NRF2-ARE signaling in microglial BV2 cells. Neurochem Int. 201 1 Feb;58(2):153-60].
  • NRF2 Nuclear Factor-like 2
  • ARE Antioxidant Responsive Element
  • XN modulates the expression of hepatic enzymes and the proteins essential for maintaining the homeostasis of the thyroid hormone (TH).
  • TH thyroid hormone
  • XN acts on the TH levels and the hepatic enzymes which are important for its breakdown and elimination [Branislav Radovic, Ragna Hussong, Clarissa Gerhauser; "Xanthohumol, a prenylated chalcone from hops, modulates hepatic expression of genes involved in thyroid hormone distribution and metabolism"; Mol. Nutr. Food Res: 54, S225-S235 (2010)].
  • XN also reduces the genotoxicity caused by reactive oxygen species and by various groups of carcinogenic mutagens ingested with food [Franziska Felk, Wolfgang W. Huber, Metka Filipic; "Xanthohumol, a prenylated flavonoid contained in beer, prevents the induction of preneoplastic lesions and DNA damage in liver and colon induced by the heterocyclic aromatic amine amino-3-methyl-imidazo[4,5-/]quinoline (IQ)"; Mutation Research: 691, 17-22 (2010)].
  • Nuclear hormone receptors are part of a very large group of transcription factors, and are efficient targets for a therapeutic strategy designed to control the metabolism of glucose, lipids, lipoproteins, bile acids, etc., whose alterations are correlated with the onset of metabolic syndrome.
  • FXR Flunesoid X Receptor
  • FXR is a member of the superfamily of nuclear hormone receptors. It regulates the synthesis of bile acids by means of a negative feedback mechanism. FXR therefore plays an important role in the metabolism of cholesterol, lipids, lipoproteins and carbohydrates. Natural ligands which act on this receptor can regulate metabolic syndrome, reducing cardiovascular risks.
  • XN can activate FXR and modulate the genes involved in the metabolism of lipids and glucose [Hajime Nozawa; "Xanthohumol, the chalcone from beer hops (Humulus lupulus L.), is the ligand for farnesoid X receptor and ameliorates lipid and glucose metabolism in KK-Ay mice”; Biochemical and Biophysical Research Communications: 336, 754-761 (2005)].
  • XN also has an immunosuppressant effect on the proliferation of T cells, the development of killer cells activated by IL-2 (LAK), cytotoxic T lymphocytes (CTL) and the production of cytokines released by the Thl helper cells (IL-2, IFN- ⁇ and TNF-a)
  • LAK IL-2
  • CTL cytotoxic T lymphocytes
  • IL-2, IFN- ⁇ and TNF-a Thl helper cells
  • XN The suppression of the cell-mediated immune response by XN is associated with inhibition of NF-kB transcription [Xiaohua Gao, Dorrah Deeb, Yongbo Liu; "Immunomodulatory activity of xanthohumol: inhibition of T cell proliferation, cell-mediated cytotoxicity and Thl cytokine production through suppression of NF-kB"; Immunopharmacol Immunotoxicol: 31, 477-484 (2009)].
  • XN exhibits a dual activity: at low concentrations it inhibits the activity of MMP-9 and elastase, and at slightly higher concentrations that of MMP-1 and MMP-2; at the same time, it stimulates the expression of type I, III and V collagen, elastin and fibrillin 1 and 2 in the fibroblasts of the dermis [M. Samuel, R. Arena, J.
  • Natural XN can be obtained by extraction from the female inflorescence of the hop plant, or synthesised. XN, some of its natural metabolites and a few synthetic analogues have so far been little studied for their biopharmacological properties [Vogel S, Heilmann J; "Synthesis, Cytotoxicity, and Antioxidative Activity of Minor Prenylated Chalcones from Humulus Lupulus”; Journal Nat. Prod: 71, 1237-41 (2008); Emily Ho, Frederik Stevens, Cristobal L. Miranda, et Al; "Prostate cancer and benign prostatic hyperplasia treatments” US 2008/0233221; R.S. Khupse, P.W. Erhardt; "Total Synthesis of Xanthohumol”; J. Nat. Prod: 70, 1507-1509 (2007)].
  • CN 101906029 discloses flavonoid derivatives with a chalcone structure and their cyclised derivatives with a 2-arylidenebenzofuranone structure as tyrosine kinase inhibitors.
  • CN 101041646 discloses 4',6'-disubstituted 2'-hydroxyl-3'-alkylaminopropyl chalcone derivatives which are useful as antitumoral agents.
  • C-prenyl and O-allyl chalcones able to inhibit the invasion of human breast cancer cells MCF7/6 in vitro are described by Mukherjee S et al., Biorg. & Med. Chem., 9(2), 337-345, 2001.
  • the present invention relates to compounds of general formula:
  • the invention also relates to the use of said compounds as medicaments, and to compositions containing them.
  • Figure 1 shows the viability of HUVEC cells evaluated with the MTT colorimetric assay.
  • Figure 2 shows the results of the chemotaxis assay conducted on HUVEC cells using Boyden chambers.
  • Figure 3 shows the results of the chemoinvasion assay conducted on HUVEC cells using Boyden chambers.
  • Figure 4 shows the tendency of HUVEC cells to organise themselves into capillary-like structures.
  • Figure 5 shows the results of the apoptosis assay conducted on HUVEC cells.
  • Figure 6 shows the results of the invasion assay conducted on HUVEC cells.
  • Figure 7 shows the results of the migration assay conducted on HUVEC cells.
  • Figure 8 contains tables summarising the results of the MTT assays.
  • the present invention relates to a compound of eneral formula (i):
  • Ri and R 2 can be, independently of one another, selected from the group comprising H; methyl; straight or branched alkyl with 2 to 10 carbon atoms; straight or branched alkyl with 2 to 10 carbon atoms containing 1 or 2 unsaturations; cycloalkyl with 4 to 6 carbon atoms; cycloalkyl with 4 to 6 carbon atoms containing 1 or 2 unsaturations; alkoxyalkyl, which can be selected from the group comprising CH 3 OCH 2 -, CH 3 OCH 2 CH 2 - or CH 3 (OCH 2 CH 2 ) n -, CH 3 (NHCH 2 CH 2 ) favor-, CH 3 (CH 2 ) n CO(NHCH 2 CH 2 ) n -, CH 3 (CH 2 ) n S0 2 (NHCH 2 CH 2 ) n -, HN(CH 2 CH 2 ) 2 N-(CH 2 CH 2 ) n -, CH 3 N(CH 2 CH 2 ) 2 N- (CH 2 CH
  • n is an integer between 1 and 5;
  • A can be a monocyclic or bicyclic aryl or a heterocyclic, aromatic or non- aromatic, monocyclic or bicyclic ring, selected from the group comprising pyrrole, pyrrolidine, 3-pyrroline, 2H-pyrrole, 2-pyrroline, indole, isoindole, 3H-indole, indolizine, indoline, carbazole, furan, benzofuran, isobenzofuran, 2H-pyran, 4H-pyran, benzo[b]thiophene, thiophene, pyridine, piperidine, 4H-quinolizine, isoquinoline, quinoline, tetrahydroquinoline, 1,8-naphthyridine, acridine, oxazole, isoxazole, benzoxazole, benzothiazole, isothiazole, thiazole, imidazole, 2-imidazole, imidazolidine, t
  • aromatic or non-aromatic heterocyclic ring can be benzocondensed and/or further substituted with halogen, alkyl, alkenyl, alkinyl, alkoxy, amino, amido, acylamido, sulphonamido, acyl, sulphonyl, aryl or heteroaryl;
  • substituents on the A ring are independently selected from the group comprising H, -O-alkyl, -OCH 3 , CI, F, Br, I, -N0 2 , -NH 2 , -NHCH 3 , -NH-alkyl, -NHCOCH 3 , -NHCO-alkyl, -NHS0 2 CH 3 , -NHS0 2 -alkyl, -S0 2 CH 3 , -S0 2 -alkyl, -S0 2 NH 2, -S0 2 NHCH 3 , -S0 2 NH-alkyl, -S0 2 NHCOCH 3 , -S0 2 NHCO-alkyl, -C0 2 H, -CONHCH 3 , -CONH-alkyl, -C0 2 CH 3 , -C0 2 -alkyl, -CONHS0 2 CH 3 and -CONHS0 2 -alkyl, alky
  • Ri, R 2 are as defined above for formula (i) and R 3 , R 4 , R 5 , R ⁇ and R 7 are selected independently from the group comprising H, -O-alkyl, -OCH3, CI, F, Br, I, -N0 2 , -NH 2 , -NHCH 3 , -NH-alkyl, -NHCOCH 3 , -NHCO-alkyl, -NHS0 2 CH 3 , -NHS0 2 -alkyl, -S0 2 CH 3 , -S0 2 -alkyl, -S0 2 NH 2, -S0 2 NHCH 3 , -S0 2 NH-alkyl, -S0 2 NHCOCH 3 , -S0 2 NHCO-alkyl, -C0 2 H, -CONHCH 3 , -CONH-alkyl, -C0 2 CH 3 , -C0 2 -alkyl, -CON
  • R 3 , R 4 , R 5 , Re and R 7 are H, and R 2 is not H or methyl.
  • A is a phenyl ring, as in the compounds of general formula (iv), (v) and (vi).
  • A is a 2-, 3- or 4-pyridyl ring.
  • Ri and R 2 are preferably, independently of one another, hydrogen or methoxymethyl.
  • R 3 , R4, R 5 , R6 and R 7 are, independently of one another, H, -OCH3, F, CI, -N0 2 , -CONHCH3, -S0 2 NH 2 , -NHSO 2 CH 3 , or the -S0 2 NHCOCH(Et)NHCOOCH 2 Ph group; wherein at least one of R 3 , R4, R 5 , Re and R 7 is H.
  • A is phenyl or 2-, 3- or 4- pyridyl;
  • K ⁇ and R 2 are, independently of one another, hydrogen or methoxymethyl, and
  • R 3 , R 4 , R 5 , R6 and R 7 are, independently of one another, H, -OCH 3 , F, CI, -N0 2 , - CONHCH3, -S0 2 NH 2 , -NHS0 2 CH 3 , or the -S0 2 NHCOCH(Et)NHCOOCH 2 Ph group; wherein at least one of R3, R 4 , R 5 , R and R 7 is H.
  • the preferred compounds according to the invention are:
  • the present invention also relates to the pharmaceutically acceptable salts and prodrugs of the compounds of general formula (i).
  • Pharmaceutically acceptable salts comprise salts with alkaline metals and salts with free bases or acids.
  • said salts can be prepared by conventional methods.
  • the pharmaceutically acceptable acids and bases used to form the salts according to the present invention can be inorganic or organic.
  • the salts, preferably metal salts can be formed with alkaline or alkaline earth metals or other salts with physiologically acceptable metals.
  • the salts can be also formed with aluminium, calcium, lithium, magnesium, potassium, sodium and zinc.
  • the preferred organic salts can be prepared from tertiary amines and quaternary ammonium salts.
  • the compounds according to the invention can also be used in the form of prodrugs, such as those obtained by reacting the compounds according to the invention containing -NH 2 or -COOH groups with alpha-amino acids or derivatives thereof protected at the amino or carboxyl group.
  • the alpha-amino acids can be either natural or non-natural.
  • An alpha-amino acid which can be used for the purposes of the present invention is N-benzyloxycarbonyl-2-aminobutyric acid.
  • the compounds according to the invention can be obtained by following the synthesis schemes described below.
  • the synthesis scheme illustrates a method of preparing compounds of general formula (i).
  • the key points of the syntheses are a Mitsunobu reaction (b), a Claisen- Schmidt condensation (c) and the removal of the methoxymethyl (MOM) protecting group (g) when Rj and/or R 2 is hydrogen.
  • the first step (a) involves partial functional isation of 2',4',6'-trihydroxyacetophenone, 1 (commercial product) with suitable halides K X and R 2 X (wherein X can be chloro or bromo or iodo), alkyl sulphates (R !
  • t-BuMe 2 SiCl from which the corresponding ether t-BuMe 2 can easily be converted to alkyl ether wherein Ki or R 2 is Me or Bn
  • diazomethane or diazoalkyls under suitable basic conditions, using alkaline or neutral carbonates or hydroxides as bases (see, for example, protection of phenols in Wuts P.G.M. and Greene T., Greene's Protective Groups in organic synthesis, 5th edition, John Wiley & Sons 2007).
  • said group must be stable under basic conditions and easily removed under mild acidity conditions, or stable under acid conditions and easily removed under mild alkalinity conditions, or stable under both acid and basic conditions but removable by catalytic hydrogenation. These delicate deprotection conditions avoid spontaneous intramolecular cyclisations which give undesirable flavone structures.
  • Step (b) (Mitsunobu reaction) allows the introduction of the prenyl group onto intermediate 2, giving prenyl ether 3 with acceptable yields.
  • phenol 2 solubilised in anhydrous THF or in a suitable anhydrous solvent, is reacted with the alcohol 3-methyl-2-buten-l -ol, diethylazadicarboxylate (DEAD) and triphenylphosphine (TPP).
  • DEAD diethylazadicarboxylate
  • TPP triphenylphosphine
  • the yields of this step may be low, in which case the reaction is conducted with phenol 2 in anhydrous toluene, using DEAD as dehydrating agent and adding triphenylphosphine and 3-methyl-2-buten-l-ol in small portions.
  • reaction times remain unchanged (about 20-24 h), the yield can be much better (up to 90-95%).
  • the reaction can also be conducted with other solvents such as dichloromethane, acetonitrile, N-methylpyrrolidinone, benzene, m- xylene and mixtures thereof.
  • solvents such as dichloromethane, acetonitrile, N-methylpyrrolidinone, benzene, m- xylene and mixtures thereof.
  • the above reaction can also use other suitable condensing agents supported on polymer resins.
  • Diisopropylazadicarboxylate (DIAD), 1, 1 '- azodicarbonyldipiperidine (ADDP), ⁇ , ⁇ , ⁇ ', ⁇ '-tetramethylazodicarboxamide (TMAD), tributylphosphine (PBu 3 ) and the like can also be used as condensing agents as well as DEAD and TPP.
  • DIAD 1, 1 '- azodicarbonyldipiperidine
  • TMAD ⁇ , ⁇ , ⁇ ', ⁇ '-tetramethylazodicarboxamide
  • PBu 3 tributylphosphine
  • the third step (c) involves a Claisen rearrangement to obtain the intermediate chalcone 4.
  • Precursor 3, solubilised in ⁇ , ⁇ -dimethylaniline, is maintained for a time of between 1 and 48 hours, preferably 4 hours, at a temperature ranging between 60 and 240°C, preferably 200°C, under stirring, to obtain the product of transposition onto the para position of aromatic ring 4 with a 44% yield when
  • the fourth step (d) consists of methylation of the hydroxyl in the ortho position (position 6') using dimethylsulphate in the presence of potassium carbonate, to give derivative 5 with yields ranging between 45 and 70%.
  • Step (f) involves aldol condensation, which leads to the protected chalcone (E)-6 (50-62%).
  • Synthesis scheme 1 (a) R ⁇ X, R 2 X, base or other; (b) 3-methyl-2-buten-l-ol, DEAD, PPh 3 , THF/toluene or other; (c) ⁇ , ⁇ -dimethylaniline, reflux, 200°C; (d) (CH 3 0) 2 S0 2 , K 2 C0 3 , acetone, reflux, 60°C; (e) mono/disubstituted arylaldehyde; (f) aqueous NaOH, MeOH, reflux, 65°C; (g) HCl/MeOH (1.25M).
  • the compounds of formula (i), wherein A is a monocyclic aryl substituted in the 4 position by a methoxyl group and in the 2 or 3 position by a fluorine atom can also be obtained by reacting the compounds of formula (i), wherein A is a monocyclic difluoro aryl substituted in the 3,4 or 2,4 positions with sodium methylate or potassium methylate, under the classic conditions used for aromatic nucleophilic substitution reactions.
  • mixtures of type (i) compounds can be obtained together with the type (ii) or (iii) cyclised compounds, and then easily separated by chromatography, n-propanol, isopropanol, n-butanol, isobutanol, cyclohexanol and various glycols can be used as alcoholic solvents, depending on the necessary conditions, in addition to methanol (MeOH).
  • the compounds according to the invention exhibit antiproliferative effects against human tumour lines, such as breast cancer, hepatocarcinoma, prostate carcinoma, myeloma and leukaemia lines.
  • the compounds according to the invention also inhibit the proliferation of human umbilical vein endothelial cells (HUVEC).
  • HBVEC human umbilical vein endothelial cells
  • the compounds according to the invention also inhibit the chemotaxis and invasion of HUVEC and human tumour cell lines, such as breast cancer and fibrosarcoma cells.
  • the compounds according to the invention therefore possess anti-angiogenic, antioxidant and chemopreventive properties.
  • the anti-proliferative, anti-invasive and anti-angiogenic activity possessed by the compounds according to the invention is greater than the activities exhibited by XN when used as control in parallel experiments.
  • the compounds according to the invention modulate the catalytic activity of extracellular matrix metalloproteases, especially between MMP-2 and MMP-9, with different selectivities from XN.
  • a further subject of the present invention is the use of the compounds according to the invention as medicaments.
  • the compounds according to the invention can be used in the prevention and/or treatment of tumoral, inflammatory, cardiovascular or neurodegenerative disorders, or as angiogenesis inhibitors, for example in the prevention and/or treatment of tumour angiogenesis.
  • the compounds according to the invention can be suitably formulated with pharmaceutically acceptable excipients or carriers.
  • suitable pharmaceutical forms can vary according to the specific compound and the administration route. The dose of active ingredient will be determined on each occasion, according to the severity of the disorder to be treated and the patient's general condition. Suitable pharmaceutical compositions can be prepared in accordance with the indications reported in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co.
  • the preparatory liquid chromatographies were conducted with flash chromatography on prepacked (Biotage) Isolute Si II columns or on columns packed by us containing 230-400 mesh silica gel.
  • the thin-layer chromatographies (TLC) were conducted with 60 F254 (MERCK) silica gel plates containing a fluorescent indicator. The various spots were highlighted with a UV lamp (256 nM). The melting points were determined under the Kofler microscope.
  • Triphenyl phosphine (1.879 g, 7.165 mmols, 1.2 equiv) and the alcohol 3-methyl- 2-buten-l-ol (0.9 mL, 8.956 mmols, 1.5 equiv) were added to a solution of 1 -(2-hydroxy- 4,6-dimethoxyphenyl)ethanone, 2 (1.530 g, 5,971 mmols, 1 equiv) in tetrahydrofuran (30 mL), placed under stirring in an ice bath. The DEAD (1.5 ml, 9.554 mmols, 1.6 equiv) was dripped and the solution was heated to RT and left under stirring for 21 h.
  • Triphenyl phosphine 2.068 g, 7.884 mmols, 1.4 equiv
  • the alcohol 3-mefhyl- 2-buten-l-ol 1.1 ml, 1 1.238 mmols, 2 equiv
  • l-(2-hydroxy- 4,6-dimethoxyphenyl)ethanone 2 (1.440 g, 5.619 mmols, 1 equiv) in toluene (29 mL)
  • the DEAD (1.8 mL, 11.238 mmols, 2 equiv) was dripped and the solution was heated to RT and left under stirring for 21 h.
  • the solvent was then evaporated and the residue suspended in ethyl ether, in which the formation of a white crystalline precipitate was observed.
  • the solid was filtered under vacuum and the filtrate evaporated, to obtain a sticky yellow oil with a pungent odour (6.260 g).
  • the crude product was purified with a flash chromatography column on silica gel (silica 230- 400 mesh, diameter 6 cm, height 17 cm) after preparation of the absorbate (silica 230-400 mesh, 7.0 g).
  • the eluent mixture used for the resolution of the column was n- hexane/AcOEt in the ratio of 7:1. 1.660 g of 3, which presented as a pale oil, was obtained from evaporation of the organic fraction (test tubes 72-123, 15 mL fractions, Vm: 750 mL).
  • the separated organic phase was dried on sodium sulphate, filtered and evaporated, to obtain a yellow oil (0.278 g).
  • the crude product was purified on an Isolute Si II lOg flash chromatography column using n- hexane/ AcOEt 12:1 as eluent. When the organic fraction had been evaporated (test tubes 25-36, 5 mL fractions), a fluorescent yellow semisolid 6 (0.163 g) was obtained.
  • Example 7 Preparation of (E)-3-(3,4-dichIoro-phenyl)-l-[2,4-dihydroxy-6- methoxy-3-(3-methyl-but-2-enyl)-phenyl]-prop-2-en-l-one [compound (8a)] (chalcone 8) and (E)-3-(3,4-dichIorophenyl)-l-(5-hydroxy-7-methoxy-2,2- dimethykhroman-6-yl)prop-2-en-l-one [compound (8b)]
  • the crude product was chromatographed on a an Isolute Si II 5 g flash chromatography column, using n-hexane/AcOEt in the ratio of 7:1 as eluent mixture.
  • 8a (0.008 g) was obtained from evaporation of the organic fraction (test tubes 5-10, 5 mL fractions) as a yellow solid.
  • 8b (0.004 g) was obtained from evaporation of test tubes 1-3 (5 mL fractions) as a yellow oil.
  • the organic phase was dried on sodium sulphate, filtered and evaporated, to obtain a yellow oil (0.183 g).
  • the crude reaction product was purified on a an Isolute Si II 10 g flash chromatography column using n-hexane/ AcOEt 14: 1 as eluent mixture. 9 (0.091 g), a bright yellow oil, was obtained from evaporation of the organic fraction (test tubes 23-27, 5 mL fractions).
  • Example 9 Preparation of (E)-l-[2,4-dihydroxy-6-methoxy-3-(3-methyI-but- 2-enyl)prenyl]-3-(4-fluorophenyl)-prop-2-en-l-one [compound (10)] and (E)-3-(4- fluorophenyl)-l-[2-hydroxy-6-methoxy-4-(methoxymethyloxy)-3-(3-methyl-but-2- enyl]-phenyl)prop-2-en-l-one [compound (11)]
  • the crude product was purified after adsorption on silica (silica 230-400 mesh, 0.080 g) by flash chromatography on silica gel column (silica 230-400 mesh, diameter 2 cm, height 15 cm) using n-hexane/EtOAc 7: 1. 1 1 (0.006 g) was obtained as an orange oil and 10 (0.021 g) as a golden yellow solid from evaporation of the organic fractions (test tubes 8-10, test tubes 16-33, 8 mL fractions).
  • the brown crude oil (0.382 g) was purified on an Isolute Si II 10 g flash chromatography column, using an n-hexane/AcOEt 6:1 eluent mixture.
  • organic fraction test tubes 20-44, 5 mL fractions
  • a yellow oil 12 (0.274 g) was obtained.
  • the crude product (0.386 g), a bright yellow oil, was purified by Isolute Si II 10 g flash chromatography using n-hexane/ AcOEt 5:1 as eluent mixture. (15) (0.090 g), which presented as a yellow oil, was obtained from evaporation of the organic fraction (test tubes 115-121, 5 mL fractions).
  • Example 13 Structural formulas of (E)-l-[2,4-dihydroxy-6-methoxy-3-(3- methylbut-2-enyI)phenyI]-3-(4-nitrophenyl)prop-2-en-l-one [compound (14)], (E)-N- (4- ⁇ 3-[2-hydroxy-6-methoxy-4-methoxymethyloxy-3-(3-methyl-but-2-enyl)phenyl]-3- oxoprop-l-enyl ⁇ phenyl)acetamide [compound (16)] and (E)-N-(4- ⁇ 3-[2,4-dihydroxy- 6-methyloxy-3-(3-methyI-but-2-enyl)phenyl]-3-oxoprop-l-enyl ⁇ -phenyl)acetamide [compound (17)]
  • the organic phase was dried on sodium sulphate, filtered and evaporated.
  • the crude product (0.434 g), a bright yellow oil, was purified after adsorption on silica (silica 230-400 mesh, 0.600 g), by flash chromatography on silica gel column (silica 230-400 mesh, diameter 3 cm, height 15 cm), using n-hexane/AcOEt 7:1 as eluent mixture. (18) (0.276 g, 0.597 mmols) was obtained as a yellow oil from evaporation of the organic fraction (test tubes 43-50, 12 mL fractions, Vm: 200 mL).
  • the organic phase was dried on sodium sulphate, filtered and evaporated.
  • the crude product (0.453 g), a bright yellow oil, was purified after adsorption on silica (silica 230-400 mesh, 0.600 g), by flash chromatography on silica gel column (silica 230-400 mesh, diameter 3 cm, height 15 cm), using n-hexane/AcOEt 8:1 as eluent mixture. (21) (0.118 g, 0.255 mmols) was obtained as a yellow oil from evaporation of the organic fraction (test tubes 56-67, 12 mL fractions, Vm: 250 mL).
  • Example 17 Preparation of (E)-3-(2,4-difluorophenyl)-l-[2-hydroxy-6- methoxy-4-methoxymethyIoxy-3-(3-methyI-but-2-enyl)-phenyI]-prop-2-en-l-one [compound (22)], (E)-3-(2,4-difluorophenyl)-l-[4-hydroxy-6-methoxy ⁇ 2- methoxymethyIoxy-3-(3-methyl-but-2-enyI)-phenyI]-prop-2-en-l-one [compound (23)] and (E)-3-(2,4-difluorophenyI)-l-[2,4-dihydroxy-6-methoxy-3-(3-methyl-but-2- enyl -phenyl]-prop-2-en-l-one [compound (24)]
  • the crude product (0.280 g), a yellow oil, was purified after adsorption on silica (silica 230-400 mesh, 0.600 g), by flash chromatography on silica gel column (silica 230-400 mesh, diameter 3 cm, height 15 cm), using n-hexane/AcOEt 2:1 as eluent mixture. Yield: 12%.
  • Example 19 Preparation of l-[2-hydroxy-6-methoxy-4-methoxymethoxy-3- (3-methyl-but-2-enyl)-phenyl]-3-pyridin-2-yl-propenone [compound (26xHCI)], l-[4- hydroxy-6-methoxy-2-methoxymethoxy-3-(3-methyl-but-2-enyl)-phenyl]-3-pyridin- 2-yl-propenone [compound (27xHCl)] and l-[2,4-dihydroxy-6-methoxy-3-(3-methyl- but-2-enyl)-phenyl]-3-pyridin-2-yl-propenone [compound (28xHCl)]
  • reaction mixture was evaporated at LP and at RT, obtaining a golden yellow crude oil (0.98 g) which was purified, after adsorption on 0.200 g of silica 230-400 mesh, by flash chromatography on silica gel column (silica 230-400 mesh, diameter 2 cm, height 15 cm) using chloroform/methanol 8:1 as eluent mixture.
  • hydrochlorides were obtained in succession by evaporation of the most significant fractions: first (26xHCl) (0.013 g) as a yellow oil, then (27xHCl) (0.022 g) as an orange-yellow oil, and finally (28xHCl) (0.011 g) as a brownish-yellow solid.
  • Example 21 Preparation of 3-(5-chloro-pyridin-3-yl)-l-[2-hydroxy-6- methoxy-4-methoxymethoxy-3-(3-methyl-but-2-enyl)-phenyI]-propenone [compound (30xHCl)], 3-(5-chloro-pyridin-3-yl)-l-[4-hydroxy-6-methoxy-2-methoxymethoxy-3- (3-methyI-but-2-enyl)-phenyl]-propenone [compound (31xHCl)] and 3-(5-chloro- pyridin-3-yl)-l-[2,4-dihydroxy-6-methoxy-3-(3-methyl-but-2-enyI)-phenyl]- propenone [compound (32xHCl)]
  • Example 23 Preparation of l-[2-hydroxy-6-methoxy-4-methoxymethoxy-3- (3-methyI-but-2-enyI)-phenyl]-3-pyridin-4-yI-propenone [compound (34xHCl)], 1- [4-hydroxy-6-methoxy-2-methoxymethoxy-3-(3-methyl-but-2-enyl)-phenyl]-3- pyridin-4-yl-propenone [compound (35xHCI)] and l-[2,4-dihydroxy-6-methoxy-3-(3- methyI-but-2-enyI)-phenyl]-3-pyridin-4-yl-propenone [compound (36xHCl)]
  • Example 25 Preparation of N-(4- ⁇ 3-[2-hydroxy-6-methoxy-4- methoxymethoxy-3-(3-methyI-but-2-enyl)-phenyl]-3-oxo-propenyl ⁇ -phenyl)- methanesulphonamide [compound (38)], N-(4- ⁇ 3-[4-hydroxy-6-methoxy-2- methoxymethoxy-3-(3-methyl-but-2-enyl)-phenyl]-3-oxo-propenyl ⁇ -phenyl)- methanesulphonamide [compound (39)] and N-(4- ⁇ 3-[2,4-dihydroxy-6-methoxy-3-(3- methyl-but-2-enyl)-phenyl]-3-oxo-propenyI ⁇ -phenyI)-methanesulphonamide
  • Example 32 Preparation of 2-chloro-5- ⁇ 3-[2-hydroxy-6-methoxy-4- methoxymethoxy-3-(3-methyl-but-2-enyl)-phenyl]-3-oxo-propenyI ⁇ - benzenesulphonamide [compound (50)], 2-chloro-5- ⁇ 3-[4-hydroxy-6-methoxy-2- methoxymethoxy-3-(3-methyl-but-2-enyl)-phenyl]-3-oxo-propenyl ⁇ - benzenesulphonamide [compound (51)], and 2-chloro-5- ⁇ 3- [2,4-dihydroxy-6- methoxy-3-(3-methyI-but-2-enyl)-phenyI]-3-oxo-propenyl ⁇ -benzenesulphonamide [compound (52)]
  • (53) is obtained by condensing (4a) with (49) under the same conditions as used for (6).
  • Example 34 Preparation of 2-chIoro-5- ⁇ 3-[2,4-dihydroxy-6-methoxy-3-(3- methyl-but-2-enyl)-phenyl]-3-oxo-propenyl ⁇ -benzenesulphonamide [compound (52)] and 2-chIoro-5-[3-(5-hydroxy-7-methoxy-2,2-dimethyl-chroman-6-yl)-3-oxo- propenylj-benzenesulphonamide [compound (54)]
  • the semisolid residue was chromato graphed on a silica gel column, eluting with a CHC13/MeOH 8:1 mixture.
  • Example 36 Preparation of [l-(2-chloro-5- ⁇ 3-[2,4-dihydroxy-6-methoxy-3-(3- methyl-but-2-enyl)-phenyI]-3-oxo-propenyl ⁇ -benzenesulphonyl-aminocarbonyl)- propyl]-benzyl-carbamate [compound (57)]
  • Compounds 60 and 63 were prepared using higher temperatures (85°C) and longer times (48 hours) than for their analogues 18 and 21.
  • Example 37 Biological activity of synthetic analogues of xanthohumol
  • the viability of the HUVEC cells was evaluated with the MTT colorimetric assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide).
  • the cells 1000/well were plated in complete medium in 96-well plates, and after complete adherence the medium was replaced with a new medium with or without the various inhibitors at different concentrations.
  • the plates were processed at different incubation times (24, 48, 72 and 96 hours), and the absorbance was measured at 570 nm.
  • the chemotaxis assay was conducted with Boyden chambers [Albini A, Iwamoto Y, Kleinman HK, Martin GR, Aaronson SA, ozlowski JM, McEwan RN. A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res. 1987 Jun 15;47(12):3239-45; Albini A, Benelli R.
  • the chemoinvasion assay a method to assess tumor and endothelial cell invasion and its modulation. Nat Protoc. 2007;2(3):504-l 1].
  • the HUVEC cells (5xl0 4 ) were pre-treated for 24 hours with the inhibitors, resuspended in serum-free medium, and plated in the upper compartment of the Boyden chamber. The complete medium was added in the lower compartment of the chamber, and used as chemoattractant.
  • the two chambers were separated by polycarbonate filters (12 ⁇ ) coated with collagen (50 ⁇ g/mL). After 6 hours' incubation at 37°C, the filters were recovered, the cells present on the upper surface of the filter were mechanically removed, and those on the lower surface were fixed in absolute ethanol and stained with DAPI. The cells were then counted in eight consecutive fields on each filter by fluorescence microscopy.
  • the chemoinvasion assay was conducted with Boyden chambers [Albini A, Iwamoto Y, Kleinman HK, Martin GR, Aaronson SA, Kozlowski JM, McEwan RN. A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res. 1987 Jun 15;47(12):3239-45; Albini A, Benelli R.
  • the chemoinvasion assay a method to assess tumor and endothelial cell invasion and its modulation. Nat Protoc. 2007;2(3):504- 11].
  • the HUVEC cells (5x10 4 ) were pre-treated for 24 hours with the inhibitors, resuspended in serum-free medium, and plated in the upper compartment of the chamber.
  • the complete medium was added in the lower compartment of the chamber, and used as chemoattractant.
  • the 12 ⁇ polycarbonate filters were coated with matrigel (1 mg/mL). After 18 hours' incubation, the cells in the upper compartment of the chamber were mechanically removed, while those adhering to the lower surface of the filter were fixed in absolute ethanol and stained with DAPI. The cells were counted double-blind in eight consecutive fields by fluorescence microscopy.
  • a 24-well plate was coated with 300 ⁇ /well of liquid matrigel (10 mg/mL) at 4°C, using cold pipettes and avoiding bubbles. The plate was then incubated for one hour at 37°C, until the matrigel polymerised.
  • the HUVEC cells, pre-treated with the different inhibitors, were resuspended in 1 niL of complete medium and then plated in the different wells.
  • the serum-free medium was used as negative control (CTRL-). After 6 hours' incubation, the organisation of the cells in capillary-like structures was examined with an inverted microscope equipped with a camera for the acquisition of images and a digital analysis system.
  • HUVEC cells tend to organise themselves into capillary-like structures when plated on a layer of matrigel, imitating in vitro the events that take place in vivo during angiogenesis. 24 hours' pre-treatment with inhibitors LR6 (10) and LR7 (11) interferes with FBS-dependent morphogenesis. Xanthohumol (XN) was used as control. The results are shown in Figure 4.
  • the HUVEC cells (lxlO 5 ) were plated in six-well plates and left to adhere for 18 hours. The next day, the cells were pre-treated with inhibitors LR6 and LR7 (10 ⁇ ) in the presence of complete medium. After 24 hours, the cells were detached, washed with PBS and transferred to tubes for cytofluorimetric analysis. The cells were pelletted and resuspended in Annexin V-binding buffer (0.01M HEPES (4-(2-hydroxyethyl)-l- piperazineethanesulphonic acid) (pH 7.4); 0.14 M NaCl; 2.5 mM CaCl 2 .
  • Annexin V-binding buffer (0.01M HEPES (4-(2-hydroxyethyl)-l- piperazineethanesulphonic acid) (pH 7.4); 0.14 M NaCl; 2.5 mM CaCl 2 .
  • Fluorescein isothiocyanate Annexin V and 7-amino-actinomycin D were added to the tubes, and the cells were incubated for 15 minutes at room temperature in the dark. The cells were then washed in PBS, the supernatant was removed and the cells resuspended in 400 mL of binding buffer. The samples were analysed on a FACSCanto cytofluorimeter (BD Biosciences), and analysed with FACSDiva Software 6.1.2.
  • the XN control at the concentration of 10 ⁇ reduces endothelial cell invasion by about 60%; some of the novel derivatives, such as LR16 and LR17, at the same concentration, exhibit a greater inhibitory effect (a reduction of about 80% and 75% respectively).
  • the inhibitory effect of the novel compounds is most evident at the concentration of 20 ⁇ .
  • the XN control at the concentration of 10 ⁇ reduces endothelial cell migration by about 50%; some of the novel derivatives, such as LR19 and LR6, at the same concentration, exhibit a greater inhibitory effect (a reduction of about 80% and 75% respectively).
  • the inhibitory effect of the novel compounds is most evident at the concentration of 20 ⁇ .

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EP14727911.1A 2013-04-08 2014-04-07 Synthetische analoga von xanthohumol Active EP2984072B1 (de)

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IT000536A ITMI20130536A1 (it) 2013-04-08 2013-04-08 Analoghi sintetici dello xantumolo
PCT/IB2014/060496 WO2014167481A1 (en) 2013-04-08 2014-04-07 Synthetic analogues of xanthohumol

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